Organoid-based single-cell spatiotemporal gene expression landscape of human embryonic development and hematopoiesis

Single-cell level characterization of embryonic development is a major benchmark of human developmental biology. Spatiotemporal analysis of stem-cell-derived embryos offers conceptual and technical advances in the field. Here, we defined the single-cell spatiotemporal gene expression landscape of human embryonic development with stem-cell-derived organoids. We established the human embryonic organoid (HEMO) from expanded potential stem cells and achieved both embryonic and extraembryonic tissues in the same organoid. Time-series single-cell RNA sequencing paired with single-cell resolution spatial revealed human embryonic development signatures such as extraembryonic placenta, yolk sac hematopoiesis neural crest, blood vessels, and cardiac mesoderm. Hematopoietic tissues eventually predominated HEMO with erythropoiesis, mekagaryopiesis, and myelopoiesis. Cell-cell communication network analysis demonstrated that trophoblast-like tissues supplied WNT signaling in neural crest cells to facilitate maturation and migration. Single-cell resolution spatial transcriptomics defined the yolk sac erythro-megakaryopoietic niche. Vitronectin-integrin signaling, a major contributor to megakaryocyte maturation, was predominant in the yolk sac niche in HEMO and to human fetal samples. Overall, our study advances the spatiotemporal analysis of human embryonic development in stem-cell-derived organoids. Highlights Modeling human embryonic development from stem cells Used of both 10X Chromium and 10X Visium to define the gene expression landscape of embryonic development and hematopoiesis WNT signaling as a regulator of neural crest maturation and EMT VTN-ITGA2B as the main contributor to Mk maturation within the yolk sac erythro-megakaryopoietic niche Graphical abstract


Dear Editor,
The cell-cell interactions are fundamental mechanisms of differentiating embryonic tissues. 1 We aimed to model human embryonic development in stem-cell-derived organoids. We exploited the time series single-cell and spatial transcriptomics to reveal the gene expression landscape in the development of embryonic tissues and their interactions.
We leveraged the Human EMbryonic Organoid (HEMO) to model the development of embryonic tissues from human pluripotent stem cells proficient in both the extra-embryonic lineage and three germ layers. 2 HEMO generation in the hematopoietic differentiation condition emulated the process of extra-embryonic lineage and three germ layers before D8, emerging hematopoiesis at D15, and predominant hematopoiesis at D18 (Fig. 1a Table 2). The flow cytometry analysis and immunofluorescence staining confirmed the hematopoietic lineages ( Supplementary  Fig. 1e, f).
We then investigated the cell-cell interactions during the development of embryonic trunk tissues by CellChat. WNT signaling from TB-like tissues was predominantly received by neural crest populations (Fig. 1h, i; Supplementary Fig. 3a, b). The high expression of neural crest specification genes (FOXD3, SOX5, SOX6, SOX9, SOX10, PAX3, PAX7, TFAP2A) and migration genes (SNAI1, SNAI2) suggested the maturation of neural crest cells ( Fig.  1j; Supplementary Fig. 3c, d). We found that ligands WNT4 and WNT6 were expressed from TB-like cells, whilst receptors LRP6 were expressed in neural crest and neuroectoderm (Fig. 1k). WNT mediators DVL, APC, and CTNNB1 lead to the expression of CCND1 and SNAI2, the latter facilitates migration of neural crest. These observations suggest a potential function of TB-like tissues in promoting neural crest maturation and migration in the HEMO.
To examine the cell-cell interactions in hematopoietic tissues, we conducted spatial transcriptomics on HEMOs at D15 when the diversity of tissue types peaked. To gain single-cell level resolution, we jointly analysed the 10X Visium transcriptomes with the matched H&E imaging by SpatialScope, a statistical method which integrates single-cell and spatial transcriptomic data to obtain the spatial distribution of the whole transcriptome at the single-cell resolution. By applying SpatialScope to our datasets, we obtained high resolution gene expression landscape of individual HEMO ( Fig. 1l; Supplementary Fig. 4a, b). We observed variability among HEMOs possibly due to different cutting layers and polarization ( Fig. 1m; Supplementary Fig. 4c).
We further examined cell-cell interactions in the HEMO #1. YSE was mainly located in the margin of HEMO, while erythroid cells and megakaryocyte lineages appeared nearby YSE. We detected the co-localization of the YSE, Ery and Mk populations in the same spots, suggesting physical cell-cell interactions. Here, we referred to these regions as the yolk sac erythro-megakaryopoietic niche (Fig. 1n, circled; Supplementary Fig. 4e). CellPhoneDB computed the spatial ligand-receptor interaction pairs between YSE, Ery, MkP and Mk. We observed a strong interactive score between YSE and Mk through vitronectin (VTN) signaling, with VTN-ITGA2B ranked highest ( Fig. 1o; Supplementary Fig. 5a, b; Supplementary Table 3). Immunofluorescence staining confirmed this niche (Supplementary Fig. 5c). VTN was exclusively expressed in YSE, while ITGA2B was expressed in the erythro-megakaryopoietic populations, with the highest expression level in Mk (Fig. 1p). Other integrin subtypes ITGB3 and ITGB5 as receptors of vitronectin were highly expressed in Mk (Fig. 1q). Notably, GFI1B and NFE2, involved in ITGB3 signaling, were also expressed in Mk. 3 Consistently, vitronectin-integrin genes revealed the similar pattern in our paired scRNA-seq dataset and spatial transcriptomics (Fig. 1r). Extracellular matrix regulates megakaryocyte maturation. 4 ITGA2B inhibition reduced megakaryopoiesis in HEMO ( Supplementary  Fig. 5d). Although there was variability among HEMOs of different cutting layers, we consistently observed the yolk sac erythromegakaryopoietic niche in difference spatial slices (Supplementary Figs. 6-9). These findings indicate that yolk sac endodermal cells are the primary source of vitronectin expression, and that the integrin pathway plays a role in promoting megakaryopoiesis in HEMOs.
Finally, we analyzed the scRNA-seq dataset of human fetal tissues (herein called 'YS hematopoiesis') in order to identify corresponding tissues to HEMOs. 5 We identified that fetal yolk sac corresponded with HEMO (Fig. 1s, t). VTN was mainly expressed in yolk sac epithelial cells and ITGA2B was mainly expressed in megakaryocytes (Fig. 1u). We defined the similar expression pattern of VTN, ITGA2B, ITGB3, ITGB5, GFI1B, and NFE2 between fetal yolk sac and HEMO (Fig. 1v YS hematopoiesis vs Fig. 1r  HEMO). HEMOs could mimic human embryonic hematopoiesis in the yolk sac, and vitronectin-integrin signaling as a molecular signature of megakaryopoiesis.

DATA AVAILABILITY
The scRNA-seq and spatial transcriptomics data reported in this study have been deposited in NCBI with the accession number PRJNA855311. The link to the dataset. The human yolk sac datasets used in this paper are public available at GSE144024. The scripts used for the analysis of scRNA-seq and spatial transcriptomics have been uploaded to GitHub: https://github.com/CHAOYiming/HEMO_analysis.